Protein therapeutics have gained significance in the past 25 years, with over 130 protein and peptide based therapeutic agents currently approved by the FDA(1-5) for treatment of a range of disease states including diabetes(6), anaemia(7) and Hepatitis C(8). Protein therapeutics may have advantages over small molecule therapeutics in that they can be highly specific and off-target effects may be minimised. Furthermore, protein based therapeutic agents may also be used in replacement therapies to restore normal function(3). However, protein and peptide based therapeutics may have risks associated with detrimental immune responses to the recombinant proteins(9), and are generally more complex and expensive to produce(10).
Almost all extracellular mammalian proteins are glycosylated(11), and the functions and physical properties of these proteins are highly dependent on the structure and level of glycosylation(12). Of the carbohydrates present in these glycans the terminal sugar is most often sialic acid(13). As the point of first contact for protein-receptor interactions, sialic acid plays an important role in the physical and biological properties of the glycoprotein. The presence of sialic acid on glycoproteins is intrinsically linked to plasma half life(14). This may be exemplified by a synthetic, hyperglycosylated erythropoiesis-stimulating agent that showed a significantly increased half-life in serum due to suppression of the clearance mechanism, mediated by the liver(15, 16), thereby effectively boosting the efficacy of the drug(7-17). The receptors in the liver that are responsible for binding to and clearing glycoproteins will often recognise a terminal galactose residue. Sialic acid is commonly found linked to galactose and so its presence on the glycan terminus effectively acts as a mask for the galactose, blocking the binding of the glycoprotein to the asialo-receptors in the liver and reducing the clearance rate.
The action of any mammalian sialidases (sialic acid hydrolysing enzymes)(18) on a glycoprotein is to remove the terminal sialic acid to expose the terminal galactose. Once this residue is exposed the protein or peptide is effectively targeted for clearance from the circulation. Thus proteins that possess glycans bearing sialidase-resistant sialosides have the potential for increased serum half-lives. This concept may be used to boost the efficacy of any synthetic, therapeutic glycoprotein or glycopeptide, provided a suitable, stable, and non-immunogenic sialic acid derivative can be found that can be readily transferred to any glycoprotein. Since spontaneous (non-enzymatic) sialoside cleavage may also be important, any substitutions on the sialic acid that minimize that process could also be of value.
A number of approaches have been described to produce sialidase-resistant sialosides, including the use of sulfur-linked sialosides(19). Replacement of the normal oxygen linkage with a sulfur rendered these S-linked sialosides far more resistant to chemical or enzymatic hydrolysis compared with their O-linked homologues. Such an approach has proven useful in providing stable sialosides to probe the binding affinities and specificities of sialic acid binding proteins(20,21), and also in antibody generation towards vaccine synthesis(22,23). Furthermore, stable sialosides have proven to be highly effective inhibitors of the sialidases of T. rangeli(24), T. cruzi(25), rotavirus(26) and influenza virus(27).
It has also been found that introduction of fluorine to the 3 position of sialic acid derivatives greatly increases the stability of the glycosidic bond. This is likely due to the close proximity of the highly electronegative fluorine atom to the anomeric carbon thereby inductively destabilizing any oxocarbenium ion-like transition state and greatly increasing the activation energy for bond cleavage. This concept has been used to great effect in the study of sialidases, by allowing trapping of a 3-fluoro sialosyl-enzyme covalent intermediate(28-30), and also of sialyltransferases, by providing a stable sialyl donor sugar (CMP 3-fluoro sialic acid 2)(31-34).